Click chemistry and in-gel fluorescence of newly synthesised proteins

Cells were mock-infected or infected with HSV by standard procedures. In control experiments, cells were incubated with HPG in the absence or presence of 100 μg/ml cycloheximide (CHX), added 1 hr before methionine depletion (S1b Fig). Cells were lysed in PBS containing 2% SDS and diluted to 1% SDS before the click reaction. 100 μg of protein samples were subjected to the click reaction as follows. Click reaction buffer consisting of the capture reagent (0.1 mM IRDye 800CW Azide Infrared Dye from LI-COR); 1 mM CuSO₄, 2 mM Tris-(2-Carboxyethyl)phosphine (TCEP; Sigma-Aldrich), 0.2 mM Tris(benzyltriazolylmethyl)amine (TBTA; Sigma-Aldrich) was freshly prepared. Following the addition of the click mixture, samples were placed on a rotating mixer for 1.5 hr at RT, and the reaction was stopped by addition of EDTA to a final concentration of 10 mM. Subsequently, proteins were precipitated (chloroform/methanol, 0.25:1, relative to the sample volume). The precipitated proteins were pelleted by centrifugation at 14,000 rpm for 5 min, washed with methanol and air dried for 10 min. The pellets were then resuspended in 1x SDS sample buffer, boiled for 5 min, and 20 μg of proteins were loaded on 12% SDS-PAGE gels. Following electrophoresis, gels were washed with water, fixed in solution containing 40% methanol, 10% acetic acid, 50% water for 5 min and washed with water. In-gel fluorescence detection of translated proteins was performed using a LI-COR Odyssey infrared imaging system, and the protein loading was assessed by Coomassie blue staining.